Chromatographic and density gradient separation of particles are utilized in biochemical research to separate macromolecules such as proteins, DNA and RNA, and larger aggregates such as enzyme complexes, ribosomes, viruses and cells. With such applications, it may be necessary to monitor the absorbance and fluorescence of particles within a flowing liquid sample, typically within a flow cell. Ultraviolet-visible spectroscopy (UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region, where absorption measures transitions from the ground state to the excited state. Fluorescence spectroscopy deals with transitions from an excited state to a ground state.
Conventional methods and apparatuses provide monitoring of the absorbance of particles in a flowing liquid. For example, one conventional flow cell (U.S. Pat. No. 8,649,005) provides an optical flow cell detector comprising a sample inlet and outlet in fluidic communication through a flow cell channel of cross sectional area A, an input light guide with a light exit surface arranged adjacent and in optical alignment with a light entrance surface of an output light guide. The input light guide and the output light guide protrude into the flow cell channel. The distance between the light exit surface and the light entrance surface is less than 1.0 mm, and the cross sectional area of the protruding portions of the input light guide and the output light guide in the flow direction is less than A/2.
As well, conventional methods and apparatuses provide monitoring of the fluorescence of particles in a flowing liquid. For example, one conventional flow cell assembly (U.S. Pat. No. 9,267,887) includes a high-pressure flow cell having a cell body made of a light-transmissive material, wherein the cell body is penetrated by a straight-line flow path for a high-pressure fluid, which allows the high-pressure fluid to be irradiated with excitation light and allows fluorescence of the high-pressure fluid to be detected.
However, neither of these flow cell designs are of use where separation of particles of interest is the goal, as it is in chromatography (e.g. FPLC and HPLC) and density gradient centrifugation. In the flow cell of U.S. Pat. No. 8,649,005, the protruding light guides may give variable path length, but they also disrupt the smooth flow of the liquid moving around them and through the gap between them. If the input liquid represents a stream of particles separated from each other by some means, the resultant turbulence, the comparatively large volume of the flow cell and the large surface area of its threaded light probes will produce smearing of the flowing liquid and a resultant loss of resolution. Additionally, this design is incompatible with fluorescence detection where a large volume of sample needs to be queried to produce a reasonable fluorescence signal.
In addition, the example fluorescence flow cells discussed above are unable to measure absorbance.
One conventional flow cell design described in U.S. Pat. No. 3,728,032 provides an oval cross section at its center, and tall, elliptical windows for optical measurements at the apices of the oval. The overall shape is one of a flattened elongated bubble having a long X-axis in the direction of the absorption light path and a much shorter Y-axis dimension. With the flow cell in U.S. Pat. No. 3,728,032, the readings are sampling an asymmetrical elongated area of a flow path. As well, the flow cell of U.S. Pat. No. 3,728,032 cannot measure fluorescence.
Another conventional flow cell design is described in U.S. Pat. No. 3,920,334 which may measure absorbance and fluorescence in applications other than density gradients. Particularly, this design views the liquid flow from outside a round glass tubing that contains the flow, which may result in a large loss of light that is compensated for with a fluorescent reflector. As well, the volume of liquid from which fluorescence and absorption signals are measured is very large, resulting in a low resolution in the flow cell which is not suitable for density gradient applications.
The only commercially available flow cell capable of both fluorescence and absorption is the PRO−FC−FL+TR (Ocean Optics, Dunedin, Fla.). However, it is designed for industrial online flow analysis applications, not for low volume separation technologies. Its adjustable path length (0.5-15 mm) indicates that it has the same disruptive interior design as the flow cell of U.S. Pat. No. 8,649,005, making it unsuitable for use with, for example, separation technologies.
One can describe three basic categories of flow cells: 1) large flow rate cells used in process technologies such as chemical production, 2) small flow rate cells like the analytical fluorescence example above, where the digital/graphic output from the flow cell is the desired result, and 3) small flow analytical cells in which the flow cell's output is informative, but the species of different particles identified in the graphic trace are processed in some way downstream of the flow cell. There is a key difference between the latter two analytical application flow cells. In the third type, the resolution achieved by the separation method (that is the source of the flow), i.e. centrifugation or chromatography, depends on fluid flow through the flow cell with as little disturbance and mixing as possible.
It would be desirable to develop an improved analytical flow cell which provides improved resolution and/or flexibility with respect to measuring light absorbance and/or emission (e.g. fluorescence).